启动子区域甲基化对肺腺癌GPC5表达的调控作用及意义

目的 研究启动子区域CpG岛的甲基化对肺腺癌细胞GPC5表达的调节作用.方法 在线预测GPC5启动子区域CpG岛的分布,合成甲基化特异性聚合酶链反应(Methylation specific polymerase chain reaction,MSP)引物,甲基化酶特异性抑制剂5-氮杂-2'-脱氧胞嘧啶核苷(5-AZA)处理肺腺癌细胞系A549、SPC-A1及永生化支气管上皮细胞HBE,检测处理前后GPC5表达的变化.同时用MSP方法检测肺腺癌组织标本中GPC5基因启动子区域CpG岛甲基化情况,以探讨其可能的临床意义.结果 GPC5在正常的支气管上皮中不表达,而在肺腺癌细胞系中均有明显表达.5-AZA处理后,GPC5在A549及SPC-A1中的表达均上调,提示启动子区域CpG岛的甲基化参与了GPC5基因表达的调节.但肺腺癌组织标本中GPC5基因启动子区域CpG岛呈现低甲基化状态,提示这种低甲基化状态参与了肺腺癌细胞中GPC5表达的上调.结论 GPC5基因启动子区域的甲基化参与了其表达的调控,可能是导致肺腺癌中GPC5上调的一个重要因素.     

Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for gamma interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5' CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell line, was treated with gamma interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma cell line, only did so when treated with 5-aza-2'-deoxycytidine followed by gamma interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of gamma interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell-lineage lymphomas.     

Downregulation of retinoic acid receptor-β2expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM:To study the role of hypermethylation in the loss of retinoic acid receptor β2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS:The role of hypermethylation in RARβ2 gene silencing in 6 ESCC cell lines was determined by methylation-specific PCR (MSP),and its methylation status was compared with RARβ2 mRNA expression by RT-PCR.The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions.RESULTS:Methylation was detected in 4 of the 6 cell lines,and the expression of RARβ2 was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2 was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ2 after 5-aza-2′-deoxycytidine (5-aza-dc) treatment.CONCLUSION:The methylation of the 5′ region may play an important role in the downregulation of RARβ2 in some ESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines.This study may have clinical applications for treatment and prevention of ESCC.     

Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

AIM： TO screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line. METHODS： Methylated DNA sequences in genome were enriched with methylated CpG islands amplification （MCA） to undergo representational difference analysis （RDA）, with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR （MSP） and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer. Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent. RESULTS： Nineteen differentially methylated sequences were obtained and located at 5＇ end, exons, introns and 3＇ end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, as the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signalsexcept with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent. CONCLUSION： Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical methods. Significant     

Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.

Mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) gene appear to be causal events for the majority of spontaneous clear-cell renal carcinomas. We now show that hypermethylation of a normally unmethylated CpG island in the 5' region provides another potentially important mechanism for inactivation of the VHL gene in a significant portion of these cancers. This hypermethylation was found in 5 of 26 (19%) tumors examined. Four of these had lost one copy of VHL while one retained two heavily methylated alleles. Four of the tumors with VHL hypermethylation had no detectable mutations, whereas one had a missense mutation in addition to hypermethylation of the single retained allele. As would be predicted for the consequence of methylation in this 5' CpG island, none of the 5 tumors expressed the VHL gene. In contrast, normal kidney and all tumors examined with inactivating VHL gene mutations but no CpG island methylation had expression. In a renal cell culture line, treatment with 5-aza-2'-deoxycytidine resulted in reexpression of the VHL gene. These findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers.     

Downregulation of retinoic acid receptor-β2expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM: To study the role of hypermethylation in the loss ofretinoic acid receptorβ2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS: The role of hypermethylation in RAR,β2 gene silencing in 6 ESCC cell lines was determined by methylationspecific PCR (MSP), and its methylation status was compared with RARβ2 mRNA expression by RT-PCR. The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions. RESULTS: Methylation was detected in 4 of the 6 cell lines, and the expression of RARβ2was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ after 5aza-2'-deoxycytidine (5-aza-dc) treatment.CONCLUSION: The methylation of the 5' region may play an important role in the downregulation of RARβ2 in someESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines. This study may have clinical applications for treatment and prevention of ESCC.     

Hedgehog通路中Ptchl基因在人胃癌中高甲基化状态分析

目的 研究Hedgehog通路中Ptchl基因表达及其甲基化状态在胃癌发生中的变化.方法 分别抽提10例胃癌组织及其癌旁＞3 cm组织和胃癌细胞株AGS的总RNA和基因组DNA.实时定量逆转录多聚酶链反应(QRT-PCR)检测Ptch基因的mRNA表达.应用软件分析Ptchl基因5'调控序列的CpG岛状态,亚硫酸氢盐测序PCR(BSP)分析甲基化水平.结果 对Ptchl mRNAla转录体转录起始位点(计为0点)上游-3950 bp和下游+2050 bp进行CpG岛分析,发现存在两个CpG岛,第1个为-1139 bp～+860 bp,第2个为+875 bp～+1692 bp.以第1个CpG岛的-870 bp～+229 bp区段内的19个CpG位点的BSP测序结果显示,胃癌细胞株AGS全部发生甲基化,胃癌组织中甲基化程度为16%～100%,平均64%±32%,癌旁组织甲基化程度为0%～42%,平均13%±14%,两组问差异有统计学意义(P＜0.05).Spearman秩相关分析发现,Ptchl基因甲基化同其表达呈负相关(r=-0.558,P=0.011).结论 Ptchl基因高甲基化参与胃癌的发生,可能为胃癌新的肿瘤标志物.     

p16基因启动子区甲基化在人嗜铬细胞瘤和副神经节瘤中的变化

目的 检测嗜铬细胞瘤(PHEO)和副神经节瘤(PGL)中p16基因突变和启动子区DNA甲基化改变,分析其与患者临床特征之间的关系.方法收集34例(PHEO 20例、PGL14例)组织标本及患者临床资料,通过甲基化特异性PCR(MSP)测定p16基因启动子区甲基化状态,DNA测序检测基因序列以及RT-PCR方法测定其mRNA表达.结果 (1)34例肿瘤组织中未发现p16基因纯合缺失及点突变;(2)35.3%(12/34)的患者存在p16基因高甲基化,p16基因甲基化阳性标本中,PHEO和PGL分别占25%和75%,两者差异有统计学意义(P=0.005);p16基因甲基化在恶性、单发肿瘤、发病年龄早的亚组中有增高趋势(P＞0.05);(3)甲基化与非甲基化肿瘤组织之间p16 mRNA表达无统计学差异;不同特点的肿瘤中其mRNA表达亦无统计学差异,但恶性肿瘤p16 mRNA表达与良性肿瘤相比有下降的趋势(0.83±0.65对1.12±0.81,P=0.278).结论人PHEO和PGL中,p16基因纯合缺失和突变并不常见,但p16基因启动子区甲基化是其失活的主要形式.     

散发性乳腺癌组织中EMSY基因启动子甲基化状态观察

目的观察散发性乳腺癌组织中EMSY基因启动子CpG岛甲基化的状态。方法用特异性甲基化PCR法检测66例散发性乳腺癌和32例正常乳腺组织中EMSY基因启动子区甲基化情况。结果乳腺癌组织有11例（16．7％）、正常乳腺组织有12例（37．5％）EMSY基因启动子发生甲基化,两者相比,P〈0．05。结论散发性乳腺癌组织中EMSY基因启动子区呈低甲基化状态,可能与散发性乳腺癌的发生发展有关。